At present, glaucoma is acknowledged as a major cause of blindness in developed countries. According to World Health Statistics 2002, the number of patients with blindness was 37,000,000 worldwide on the basis of the WHO standard, and 12% of such patients (i.e., 4,500,000 patients) suffer from glaucoma-induced blindness. In Japan, about 20% of visual impairment cases are caused by glaucoma, which is the leading cause of blindness. According to survey results, in addition, about 5% of the population aged 40 and older has been afflicted with glaucoma in Japan. It is also known that the prevalence rate of glaucoma increases with age. In the years to come after the arrival of an aging society, accordingly, the number of patients is deduced to be increasing. Since the clinical condition of glaucoma is retinal ganglion cell death caused by optic-disc cupping, treatment thereof is concentrated on the protection of retinal ganglion cells. The initial means for retinal ganglion cell protection is to lower intraocular pressure, and lowering of intraocular pressure with the use of eye drops is the first option for glaucoma treatment. While eye drops are effective for the lowering of intraocular pressure to a certain extent, the lowering of intraocular pressure is often insufficient to stop the progression of glaucoma. In addition, eye drops merely prevent progression, and use of eye drops would not lead to radical treatment.
Accordingly, there is urgent need for the establishment of cell replacement therapy that enables radical treatment of ocular diseases caused by retinal ganglion cell death, such as glaucoma, by preparing retinal ganglion cells and replacing the dead retinal ganglion cells with the prepared retinal ganglion cells. In the field of regenerative medical techniques that include such cell replacement therapy, the advancement in stem cell engineering has enabled induction of ocular tissues or cells, such as retina and crystalline lens, from mouse or human ES or iPS cells. In the future, treatment of ocular diseases, including glaucoma, which were not possible to effectively treat in the past, is expected to become possible through implantation of such induced ocular tissues or cells.
In the past, several attempts had been made to induce pluripotent stem cells to differentiate into retinal tissues. For example, Non-Patent Document 1 describes that three-dimensional culture of human ES cells had led to regeneration of all layers of the retina. Non-Patent Document 1 describes that retinal ganglion cells were induced from human ES cells 30 days after the initiation of induction, retinal ganglion cells were identified in the inner layer of the retina as a result of immunostaining of Brn3b, which is a retinal ganglion cell marker, and the neuroepithelial cell of the retina was induced when culture was continued for 100 or more days. While all layers of the retina were successfully regenerated according to the method described in Non-Patent Document 1, axons connected to the retinal ganglion cells were not elongated. Non-Patent Document 2 describes a method of inducing retinal progenitor cells serving as cellular components constituting the retina by repeatedly subjecting iPS cells to three-dimensional culture and two-dimensional culture, preparing optic-vesicle-like structures, and continuously subjecting the prepared optic-vesicle-like structures to three-dimensional floating culture. According to the method described in Non-Patent Document 2, however, a retinal layer structure is not formed, the possibility of successfully inducing cellular components constituting the retina is low, and axons of retinal ganglion cells have not resulted in elongation. Non-Patent Document 1 describes a method for producing retinal-layer-specific neurons by allowing Notch signal pathway inhibitors to react with the retinal progenitor cells included in the retinal tissue induced to differentiate from the pluripotent stem cells via floating culture. However, although some cells observed in the produced retinal tissue were ganglion cells, a majority of such cells were visual cells.
When practicing regenerative medicine, completeness of the cells should be first taken into consideration. Even if the results of immunostaining on the retinal ganglion cell-specific markers are positive and expression of such marker genes is observed, it is highly unlikely that such cells would be applicable to treatment without axon elongation. That is, clinically applicable retinal nerve fiber layers with elongated axons have not yet been regenerated according to conventional techniques. In addition, such techniques are not practically satisfactory in terms of the rate of induction or the number of days until induction.
In the development of an agent for treatment of optic neuropathy of the retina represented by glaucoma, animal models of diseases, such as mouse models of hereditary high-tension glaucoma (DBA/2J mice), mouse models of drug-induced disorders, various gene knock-out mice, and mutant mice, have been established. However, such disease animal models are disadvantageous since breeding and management thereof are laborious, animal cells are significantly different from human cells, and evaluation of therapeutic effects of drugs may not be definite.